Regulation of Paramecium primaurelia glycosylphosphatidyl-inositol biosynthesis via dolichol phosphate mannose synthesis

• Published in: Biochimie 2001-08, Vol.83 (8), p.801-809
• Main Authors: Azzouz, Nahid, Gerold, Peter, Kedees, Mamdouh H., Shams-Eldin, Hosam, Werner, Regina, Capdeville, Yvonne, Schwarz, Ralph T.
• Format: Article
• Language: English
• Subjects: Animals; Dol-P-man; dolichol; dolichol monophosphate; Dolichol Monophosphate Mannose - metabolism; glycosylphosphatidyl-inositol; Glycosylphosphatidylinositols - biosynthesis; GTP-Binding Protein alpha Subunits, Gi-Go - metabolism; GTP-Binding Protein alpha Subunits, Gs - metabolism; Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology; Mannosyltransferases - antagonists & inhibitors; Paramecium - metabolism; Paramecium primaurelia; Phosphotransferases (Alcohol Group Acceptor) - antagonists & inhibitors
• ISSN: 0300-9084; 1638-6183
• DOI: 10.1016/S0300-9084(01)01317-7

A set of glycosylinositol-phosphoceramides, belonging to a family of glycosylphosphatidyl-inositols (GPIs) synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia has been described. The final GPI precursor was identified and structurally characterized as: ethanolamine-phosphate-6Manα1-2Manα1-6(mannosylphosphate) Manα1-4glucosamine-inositol-phospho-ceramide. During our investigations on the biosynthesis of the acid-labile modification, the additional mannosyl phosphate substitution, we observed that the use of the nucleotide triphosphate analogue GTPγS (guanosine 5ˈ- O-(thiotriphosphate)) blocks the biosynthesis of the mannosylated GPI glycolipids. We show that GTPγS inhibits the synthesis of dolichol-phosphate-mannose, which is the donor of the mannose residues for GPI biosynthesis. Therefore, we investigated the role of GTP binding regulatory ‘G’ proteins using cholera and pertussis toxins and an intracellular second messenger cAMP analogue, 8-bromo-cAMP. All the data obtained suggest the involvement of classical heterotrimeric G proteins in the regulation of GPI-anchor biosynthesis through dolichol-phosphate-mannose synthesis via the activation of adenylyl cyclase and protein phosphorylation. Furthermore, our data suggest that GTPγS interferes with synthesis of dolichol monophosphate, indicating that the dolichol kinase is regulated by the heterotrimeric G proteins.